Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells

7H2HM is a new humanized recombinant monoclonal antibody (MAb) directed against insulin-like growth factor-1 receptor and produced in CHO cells. Homogeneity of intact antibody, reduced light and heavy chains, Fab and Fc fragments were investigated by analytical methods based on mass (SDS-PAGE, SEC), charge (IEF, C-IEX) and hydrophobicity differences (RP-HPLC, HIC) and compared side-by-side with A2CHM, produced in NS0 cells. Primary structures and disulfide bridge pairing were analyzed by microsequencing (Edman degradation), mass spectrometry (MALDI–TOF, ES–TOF) and peptide mapping after enzymatic digestion (Trypsin, endoprotease Lys-C, papain). The light chains demonstrated the expected sequences. The heavy chains yielded post-translational modifications previously reported for other recombinant humanized or human IgG1 such as N-terminal pyroglutamic acid, C-terminal lysine clipping and N-glycosylation for asparagine 297. More surprisingly, two-thirds of the 7H2HM heavy chains were shown to contain an additional 24-amino-acid sequence, corresponding to the translation of an intron located between the variable and the constant domains. Taken together these data suggest that 7H2HM is a mixture of three families of antibodies corresponding (i) to the expected structure (17%; 149 297 Da; 1330 amino acids), (ii) a variant with a translated intron in one heavy chains (33%; 152 878 Da; 1354 amino acids) and (iii) a variant with translated introns in two heavy chains (50%; 154 459 Da; 1378 amino acids), respectively. RP-HPLC is not a commonly used chromatographic method to assess purity of monoclonal antibodies but unlike to SEC and SDS-PAGE, was able to show and to quantify the family of structures present in 7H2HM, which were also identified by peptide mapping, mass spectrometry and microsequencing

Analysis of antibody-drug conjugates by comprehensive on-line two-dimensional hydrophobic interaction chromatography x reversed phase liquid chromatography hyphenated to high resolution mass spectrometry. II- Identification of sub-units for the characterization of even and odd load drug species

Sabine Heinisch and Morgan Sarrut wish to thank Christelle Margoum and Philippe Bados (IRSTEA, France) for the loan of their Waters Q-Tof instrument. They also want to thank Waters for the generous gift of the 2D-ICLASS instrument and for making Maxent software available in their Lab.International audienc

Analysis of antibody-drug conjugates by comprehensive on-line two-dimensional hydrophobic interaction chromatography x reversed phase liquid chromatography hyphenated to high resolution mass spectrometry. I − Optimization of separation conditions

Antibody-drug-conjugates (ADCs) manufacturing leads to a mixture of species which needs to be char-acterized during development and for further quality control. The coupling of on-line HIC x RPLC tohigh resolution mass spectrometry can be considered as a very efficient analytical method, providingextensive information on ADC sample, within a reduced time scale. Our intention in this first paper is topresent the approach used to rationally optimize the numerous conditions that can affect the quality ofthe 2D-separation. HIC and RPLC conditions were therefore optimized to prevent salt precipitation due tosolvent mixing and to enhance sensitivity, while limiting the total analysis time. We demonstrated thatadding salt in the sample solvent before HIC injection allows a significant peak shape improvement. Thegradient profile was also carefully optimized in both dimensions, leading to a two-step gradient in HICand bracketed gradient in RPLC. This study shows that on-line HIC x RPLC hyphenated to high resolutionmass spectrometry is a useful method to obtain rapid and extensive structural information on the peaksobserved in the first HIC dimension, thereby leading, in a single step requiring 75 min, to the precisedetermination of the average drug-to-antibody ratio (DAR) by HIC as well as the knowledge of the drugload distribution for a particular DAR. The structural characterization of ADC fragments by RPLC-QTOFwill be discussed in the second part of this two-part series

Cutting-edge mass spectrometry methods for the multi-level structural characterization of antibody-drug conjugates

International audienceAntibody drug conjugates (ADCs) are highly cytotoxic drugs covalently attached via conditionally stable linkers to monoclonal antibodies (mAbs) and are among the most promising next-generation empowered biologics for cancer treatment. ADCs are more complex than naked mAbs, as the heterogeneity of the conjugates adds to the inherent microvariability of the biomolecules. The development and optimization of ADCs rely on improving their analytical and bioanalytical characterization by assessing several critical quality attributes, namely the distribution and position of the drug, the amount of naked antibody, the average drug to antibody ratio, and the residual druglinker and related product proportions. Here brentuximab vedotin (Adcetris®) and trastuzumab emtansine (Kadcyla®), the first and gold-standard hinge-cysteine and lysine drug conjugates, respectively, were chosen to develop new mass spectrometry (MS) methods and to improve multiplelevel structural assessment protocols

UV and X-ray structural studies of a 101-residue long Tat protein from a HIV-1 primary isolate and of its mutated, detoxified, vaccine candidate.

International audienceThe 101-residue long Tat protein of primary isolate 133 of the human immunodeficiency virus type 1 (HIV-1), wt-Tat(133) displays a high transactivation activity in vitro, whereas the mutant thereof, STLA-Tat(133), a vaccine candidate for HIV-1, has none. These two proteins were chemically synthesized and their biological activity was validated. Their structural properties were characterized using circular dichroism (CD), fluorescence emission, gel filtration, dynamic light scattering, and small angle X-ray scattering (SAXS) techniques. SAXS studies revealed that both proteins were extended and belong to the family of intrinsically unstructured proteins. CD measurements showed that wt-Tat(133) or STLA-Tat(133) underwent limited structural rearrangements when complexed with specific fragments of antibodies. Crystallization trials have been performed on the two forms, assuming that the Tat(133) proteins might have a better propensity to fold in supersaturated conditions, and small crystals have been obtained. These results suggest that biologically active Tat protein is natively unfolded and requires only a limited gain of structure for its function

Fusion-negative Rhabdomyosarcoma 3D-organoids as an innovative model to predict resistance to cell death inducers

Rhabdomyosarcoma (RMS) is the main form of soft-tissue sarcoma in children and adolescents. For 20 years, and despite international clinical trials, its cure rate has not really improved, and remains stuck at 20% in case of relapse. The definition of new effective therapeutic combinations is hampered by the lack of reliable models, which complicate the transposition of promising results obtained in pre-clinical studies into efficient solutions for young patients. Inter-patient heterogeneity, particularly in the so-called fusion-negative group (FNRMS), adds an additional level of difficulty in optimizing the clinical management of children and adolescents with RMS. Here, we describe an original 3D-organoid model derived from relapsed FNRMS and show that it finely mimics the characteristics of the original tumor, including inter- and intra-tumoral heterogeneity. Moreover, we have established the proof-of-concept of their preclinical potential by re-evaluating the therapeutic opportunities of targeting apoptosis in FNRMS from a streamlined approach based on the exploitation of bulk and single-cell omics data

Fusion-negative rhabdomyosarcoma 3D organoids to predict effective drug combinations: A proof-of-concept on cell death inducers

International audienceHighlights d Relapsed rhabdomyosarcoma (RMS) 3D organoids can be derived from needle biopsies d RMS 3D organoids finely preserve inter-and intra-tumor heterogeneity d Transcriptomic approach reveals Survivin as a key apoptosis blockage point in RMS d RMS 3D organoids are powerful tools to design and/or reexplore drug combinations Author